The effects of GITR-deficiency on immunity to LCMV are largely CD4 T cell-intrinsic.

<p>(A) Schematic indicating that lethally irradiated age- and sex-matched C57BL/6 mice were reconstituted with a 1:1 mixture of CD45.2 GITR^+/+: CD45.1 GITR^-/- bone marrow cells. (B) At 90 days post-reconstitution, mice were bled to evaluate the proportion of GITR^+/+ (CD45.2) and GITR^-/- (CD45.1) total, CD8, CD4, and CD4 Foxp3⁺ cells prior to infection as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004517#ppat.1004517.g001" target="_blank">Fig. 1</a>. (C, D) The proportions of GITR^+/+ and GITR^-/- total CD4, Foxp3⁺ Treg, and CXCR5⁺ PD-1^hi Tfh were determined at day eight p.i., with representative staining shown for Tfh. (E, F) The proportions of IFNγ⁺ or IFNγ⁺IL-2⁺ GITR^+/+ and GITR^-/- CD4 T cells were evaluated following five hours of I-A^b-restricted LCMV GP_61–80 peptide restimulation, with representative staining shown. (G, H) The frequencies of GITR^+/+ and GITR^-/- D^b/NP_396–404- and D^b/GP_33–41-specific CD8 T cells, and the expression of PD-1 and Tim-3 in the spleen were evaluated at day eight p.i. (I, J) Freshly isolated splenocytes from chimeric mice sacrificed at day eight p.i. were restimulated with D^b-restricted LCMV GP_33–41 peptide for five hours in the presence of Brefeldin A and monensin, after which intracellular cytokines and surface markers were measured by flow cytometry, with representative staining shown. (K, L) Co-production of TNF and CD107a by the IFNγ⁺ population was evaluated, with representative staining shown. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two experiments with a total of ten mice, for all parameters except NP_396–404, which was only evaluated in one experiment of four mice.